Gas chromatography (GC) is the chemical analysis technique most widely used to separate and identify gas components, and it has been extensively applied in various gas analysis fields such as non-invasive medical diagnoses, indoor air quality monitoring, and outdoor environmental monitoring. Micro-electro-mechanical systems (MEMS)-based GC columns are essential for miniaturizing an integrated gas analysis system (Micro GC system). This study reports an open-tubular-type micro GC (μ-GC) column with internal bump structures (bump structure μ-GC column) that substantially increase the interaction between the gas mixture and a stationary phase. The developed bump structure μ-GC column, which was fabricated on a 2 cm × 2 cm μ-GC chip and coated with a non-polar stationary phase, is 1.5 m-long, 150 μm-wide, and 400 μm-deep. It has an internal microfluidic channel in which the bumps, which are 150 μm diameter half-circles, are alternatingly disposed to face each other on the surface of the microchannel. The fabricated bump structure μ-GC column yielded a height-equivalent-to-a-theoretical-plate (HETP) of 0.009 cm  at an optimal carrier gas velocity of 17 cm/s. The mechanically robust bump structure μ-GC column proposed in this study achieved higher separation efficiency than a commercially available GC column and a typical μ-GC column with internal post structures classified as a semi-packed-type column. The experimental results demonstrate that the developed bump structure μ-GC column can separate a gas mixture completely, with excellent separation resolution for formaldehyde, benzene, toluene, ethylbenzene, and xylene mixture, under programmed operating temperatures. The analysis of volatile organic compounds (VOCs) is fundamental in a wide range of applications, including public safety management, non-invasive medical diagnoses, indoor air quality monitoring, and outdoor environmental monitoring ,6,7,8,9.Miniaturization of gas chromatographic systems provides numerous advantages. A typical GC system with reduced size affects the kinetics of the exchanges by decreasing the distances the solutes must diffuse during mass transfer between the mobile and stationary phases ,18. Sincμ-GC columns are generally classified into open-tubular and packed types, as shown in μ-GC columns that feature a micro-post array along the length of the column channel, referred to as semi-packed-type columns c have reIn this paper, we demonstrate a mechanically robust bump structure μ-GC column with a comparable separation performance. The μ-GC column was fabricated through the MEMS processes. The fabrication flow of the bump structure μ-GC column is shown in For comparison of the separation performance, the post structure μ-GC column was fabricated through the same micromachining processes previously described. The circular post structures formed in the semi-packed-type column channel exhibited a diameter of 30 μm and were disposed at intervals of 150 μm along the microchannel.The MEMS-based GC columns were commonly fabricated on 2 cm × 2 cm μ-GC chips. The column channels were 1.5 m-long, 150 μm-wide, and 400 μm-deep and were coated with a non-polar stationary phase in the form of a thin-film.The stationary phase is the key component of the GC column. It reacts with the analytes and affects the rate of the diffusion in and out of the mobile phase. The non-polar stationary phase used in this study primarily interacts with analytes by the van der Waals force. Analytes with higher molecular weights generally have a stronger interaction with the stationary phase and demonstrate longer retention times.After the channel was sealed, the stationary phase was usually applied via the liquid coating method. Static coating was the most frequently used method, and a non-polar stationary phase was generally used for the separation column. In the static coating procedure, the GC column was filled with the stationary-phase solution; one end of the column was sealed, and the other end was connected to a vacuum pump. The solvent was slowly evaporated by the reduced pressure of the vacuum pump until all of the solvent was completely evaporated; the coating was then complete. The static coating allows a uniform distribution of films because of the lack of axial motion of the stationary phase during deposition . The nonThe experimental setup is illustrated in On the other hand, the temperature is an important factor that affects the separation performance of the column. In a GC column, there is an equilibrium of the analyte between the mobile phase and the stationary phase, which is affected by the column temperature. When the separation is performed under isothermal conditions at low temperatures, the weakly retained compounds are adequately separated while strongly retained compounds demonstrate severe tailing and long retention times. Conversely, a separation performed under higher isothermal conditions leads to excellent separation of strongly retained compounds but results in poor separation of weakly retained compounds. This general elution issues can be solved by programming the temperature of the GC column . Therefo1-C12 hydrocarbons as described in [The temperature of the μ-GC column was maintained at 30 °C for 1 min and ramped to 90 °C at a ramping rate of 15 °C/min and maintained at 90 °C for 1 min. The electric power values from 1.1 W to 3.3 W were used for temperature ramping from 30 °C to 90 °C. The gas components separated through the μ-GC column were detected with a commercial FID , and the separation results were collected through a data acquisition system. Separation performance tests were carried out for both of the fabricated μ-GC columns under the same experimental conditions, and the results were compared. On the other hand, the commercial GC column with a bonded-porous-silica-based stationary phase  was prepared by being cut to a length of 1.5 m (320 μm I.D.), which is the same length as the μ-GC columns, for the comparison of the gas separation efficiency based on the Golay equation. This commercial GC column is specified for the application to the separation of Cribed in ,56.gD and sD are the binary diffusion coefficients in the mobile and stationary phases, respectively; ū is the velocity of the carrier gas; and f1 (varies between 1 and 1.125) and f2 (varies between zero and one) are the Gidding-Golay and Martin-James gas compression coefficients, respectively; w and h are the width and height of the column channel, respectively; k and fd are the retention factor and stationary phase thickness, respectively. The smaller the HETP value for a given column length, the higher the separation efficiency of the column. As a very thin thickness of the stationary phase film is assumed, the contribution of band-broadening due to diffusion in the stationary phase can be neglected. Therefore, the 3rd term in Equation (1) can be ignored to simplify the calculation [n-hexane was used to determine the average carrier gas velocity for calculating the corresponding HETP value of the GC columns. In addition, effective widths of 75 μm and 120 μm for the bump and post structure μ-GC columns, respectively, were used in the calculations. The value of gD was chosen as 0.076 cm2/s [The separation efficiency of a GC column can be defined by the height-equivalent-to-a-theoretical-plate (HETP), expressed in Equation (1) .(1)HETPculation . An n-he76 cm2/s . Figure 76 cm2/s ,60,61. OX-axis shows the time (min), the left Y-axis shows the FID detection signal level (mV), and the right Y-axis shows the column temperature (°C). The temperature of the column was maintained at 30 °C and 90 °C for 1 min, respectively, to stabilize the FID signal at the start and end of the analysis. The mixture sample of FBTEX was injected at the 1-min point after the beginning of the analysis. Subsequently, the gas separation was started with the injection of nitrogen, which is an inert gas and does not react with the FID, as a carrier gas at a flow rate ranging from 0.3 sccm to 0.4 sccm into the μ-GC column.Separation performance tests were conducted using the fabricated bump structure μ-GC column and post structure μ-GC column for the FBTEX mixture gas under the same analytical conditions. The commercial FID detected the gas components separated through the μ-GC columns, and the results were compared and analyzed. sR) is expressed in Equation (2) [rt and bw are the retention time and the peak width at the base, respectively. The data for the retention time (rt) and peak width (bw) for the separated individual gas components presented in The separation resolution was also calculated as the separation performance index of the columns. The separation resolution (tion (2) ,16.(2)RsR values for the F, B, T, E, and X gases based on the separation results obtained with the μ-GC columns. The sR values for the post structure μ-GC column between F and B, between B and T, between T and E, and between E and X were 0.63, 2.31, 0.52, and 1.24, respectively. By contrast, the sR values for the bump structure μ-GC column between F and B, between B and T, between T and E, and between E and X were 2.22, 5.19, 0.87, and 1.77, respectively, thus presenting sR values greater than those of the semi-packed-type column.The bump structure μ-GC column showed excellent separation efficiency and performance, resulting in higher separation resolutions compared with the post structure μ-GC column.We considered that the bump structure μ-GC column has sufficient sample capacity and separation efficiency because of the larger surface area created by the bump structures in the column channel. For the semi-packed-type μ-GC column with a high aspect ratio micro-post array embedded in the column channel, the micro-post array can be easily collapsed and distorted during the microfabrication and stationary-phase coating processes of the column. Furthermore, if the micro-post structures are excessively dense, thereby enabling an increase in the sample capacity, a massive pressure drop may occur inside the column channel, resulting in the channel clogging at the defect area and a reduction of the separation efficiency. The bump structure μ-GC column proposed in this study is mechanically robust compared with the conventional semi-packed-type μ-GC column and exhibits excellent separation performance with higher separation resolution under the same column length and separation experimental conditions.This study reports a mechanically robust bump structure μ-GC column with a comparable separation performance. The μ-GC column has an internal microfluidic channel wherein the bump structures are alternately disposed to face each other on the surface of the microchannel. It was fabricated on a 2 cm × 2 cm size μ-GC chip and coated with the OV-1 thin-film stationary phase, which has a length of 1.5 m, a width of 150 μm, and a depth of 400 μm. Based on the Golay equation, the separation efficacy of the developed bump structure μ-GC column was compared with the post structure μ-GC column and commercial column. We found that the fabricated bump structure μ-GC column yielded a HETP of 0.009 cm  at an optimal carrier gas velocity of 17 cm/s. The post structure μ-GC column and commercial GC column had the HETP values of 0.014 cm (7140 plates/m) and 0.043 cm (2330 plates/m), respectively. The developed μ-GC column showed about 1.6 times higher separation efficiency than the post structure μ-GC column in the comparison of HETP values. In addition, the plate number of the bump structure μ-GC column is comparable to the reported plate number of 4200~12,800 plates/m in other recent studies related to MEMS-based columns with similar physical dimensions. However, the developed bump structure μ-GC column is mechanically robust compared to the conventional post structure μ-GC column reported in other literatures.The separation performance tests of the fabricated μ-GC columns were performed using the commercial FID system for the FBTEX mixture gas known as representative hazardous VOCs. The developed bump structure μ-GC column showed higher separation efficiency than the post structure μ-GC column. The developed column can be used in various applications requiring analyses of complex gas mixtures, such as non-invasive medical diagnoses, indoor air quality monitoring, and outdoor environmental monitoring.


Background: Urothelial carcinoma associated 1 (UCA1), a novel long noncoding RNA (lncRNA) which is first discovered in 2006 in human bladder cancer and has become a hot spot in recent years. UCA1 has been demonstrated correlated with clinical outcomes in various cancers. However, the results from each study are insufficient and not completely consistent. Therefore, we perform a systematic meta-analysis to evaluate the value for a feasible biomarker for metastasis and prognosis of cancer. Methods: Relevant English literatures were searched in PubMed, Cochrane Library, Web of science, Embase databases and Chinese literatures were searched in Chinese National Knowledge Infrastructure Wanfang from inception up to 17 April 2018. The pooled odds ratio (OR) and hazard ratio (HR) with 95% confidence interval (CI) using random/fixed-effect were used to identify the relationship between UCA1 and lymph node metastasis (LNM) or overall survival (OS) of cancer patients. Subgroup analysis and sensitivity analysis were performed. The current meta-analysis was performed using Review Manager 5.3 and Stata 12.0 software. Results: A total of 3411 patients from 38 studies were finally included. Patients who with high UCA1 expression suffered from an increased risk of LNM . UCA1 was also significantly associated with OS . Subgroup analyses across several different variables also showed the similar results in LNM and OS of cancer patients. Conclusion: High expression of UCA1 was linked with poor clinical outcome. UCA1 can serve as a potential molecular marker for metastasis and prognosis in different types of cancers. Cancer is a major global public health problem that seriously threatens human health. In recent years, the incidence and mortality of cancer are also increasing year by year. According to GLOBCAN 2012, there were 14.1 million new cancer cases, 8.2 million cancer deaths and 32.6 million people living with cancer (within 5 years of diagnosis) in 2012 worldwide . However, when conducting subgroup analyses on tumor type, we found no significant correlation between high UCA1 expression and LNM among the studies in respiratory system . According to the results presented in P<0.00001) and β-actin group , while no significant association in RNU6B/GUSB group .Subgroup analyses across several different variables were further performed to investigate the heterogeneity of the studies for meta-analysis of UCA1 and LNM or OS. The LNM-related data were stratified into subgroups based on sample size, tumor type, cut-off value and reference control. The assessment results in each subgroup are also shown in P=0.06). As for different race for UCA1, the relationship between UCA1 expression and OS was significant in Asian group , but not in others group .The OS-related data were stratified into subgroups based on sample size, tumor type, cut-off value, follow-up time, analysis method, race and reference control. The detailed assessment results in each subgroup are also shown in Multiple sensitivity analyses were carried out to evaluate whether individual study influenced pooled ORs or HRs by excluding one study by turns. It was found that none of the exclusions of a specific study would change the magnitude or direction of the summary effect for the correlation between UCA1 expression and LNM or OS, which further confirmed the validity of the results  and 5.t = 1.31, P=0.202)   . However P<0.05) . Because P<0.05) . While tEvidence from multiple publications demonstrated that lncRNAs, similar to protein-coding genes, can act as oncogenes or tumor suppressor genes, which involving in a variety of tumorigenesis processes, including proliferation, invasion, migration and apoptosis. With the rapid development of high-throughput genome-wide analysis technology, more and more functional lncRNAs have been found to have potential value on predicting cancer progression. UCA1, a novel functional lncRNA which was first discovered in 2006 in human bladder cancer. In recent years, a growing number of studies have shown that UCA1 up-regulated in several cancers, including HCC, GC and lung cancer . UCA1 inIn the present meta-analysis, we systematically explore the relationship between UCA1 and cancer metastasis or prognosis. The results of the current study demonstrated that high UCA1 expression level was positively related to increasing the risk of LNM in cancer patients. Moreover, we also identified that there was a significantly positive correlation between high UCA1 expression and short OS in cancer patients. In multiple sensitivity analyses, we did not detect any substantial difference in pooled estimates, and there was no excessive influence on the overall results in any individual study.miR-204-5p, miR-193a-3p, miR-145, miR-143, miR-216b, miR-203, miR-196a-5p and miR-135a in several different cancers. For example, Zhang et al. found that UCA1 could directly interacted with miR-204 and functioned as a ceRNA, thus regulating the expression of ATF2 and promoting cell proliferation and metastasis in prostate cancer [miR-193a-3p target gene ERBB4 by competitively ‘spongeing’ miR-193a-3p in NSCLC [miR-145 and its target gene the actin-binding protein fascin homologue 1 (FSCN1) [miR-216b directly and down-regulated the expression of miR-216b. UCA1 reversed the inhibitory effect on the growth and metastasis of miR-216b of HCC, which might be involved in the suppression of fibroblast growth factor receptor 1 (FGFR1) expression, a target gene of miR-216b, and the activation of ERK signaling pathway [miR-203 effectively and thus activating the expression of transcription factor Snail2 and promote HCC progression [miR-143 [miR-196a-5p expression by binding with its promoter and thereby modulating the influence on cisplatin/gemcitabine resistance [miR-182 dependent iASPP regulation [miR-216b and activating FGFR1/ERK signaling pathway [The exact mechanisms underlying the association between elevated UCA1 expression and more LNM or poor prognosis is poorly understood, and the related reports are not the same, but many similarities were still existed. Several literatures have suggested potential mechanisms that could be involved in the metastasis and prognostic impact of UCA1 on carcinogenesis. First, UCA1 could act as a key competing endogenous RNA (ceRNA) or sponge for in NSCLC . In blad (FSCN1) . In HCC, pathway . In addigression . In blad[miR-143 . UCA1-acsistance . Second,sistance ,52. Silesistance . Fan et sistance . Third, sistance . Mechani pathway . SimultaOtherwise, some limitations to this meta-analysis should be taken into account. First, the cut-off values of UCA1 high and low expression were lack of uniform standard due to different methods and criteria in different types of cancer, which may result in some heterogeneity and affect the results of the study. Second, most studies tended to report positive results rather than negative results; our meta-analysis may overestimate the significance of UCA1 to some extent. Third, some of the HRs were estimated from survival curves rather than directly obtained from the primary studies. Lastly, most of the included studies were performed in the population from Asian countries rather than worldwide population; our results should be substantiated by additional studies in other races. Although there are some limitations, but this current meta-analysis still has its noteworthy advantages. First, 38 literatures which including a total of 3411 cases and 19 types of cancer were included in this meta-analysis. The sample size included was the largest, which significantly improved the statistical efficiency and accuracy of the test. Second, the number of search databases were greater and cancer types were more comprehensive in this meta-analysis compared with previous reports. Finally, the inclusion and exclusion criteria were more stringent and the quality of the literatures incorporated was higher.In conclusion, even some limitations mentioned above, our meta-analysis reveals that the expression level of UCA1 was significantly associated with metastasis and prognosis in different types of cancer. The higher expression of UCA1, the higher probability of occurrence of LNM cancer patients suffer with. Meanwhile, shorter OS may be observed in the patients with high UCA1 expression. Thus, UCA1 might be a novel predictive marker for estimating the metastasis and prognosis in different types of cancer. However, the significance of UCA1 in LNM in respiratory system cancers and RNU6B/GUSB reference control group should incorporate more studies to validate this result, and so does in urinary system prognosis and non-Asian people prognosis.


Due to the similar polarity and strong interaction between LMPET and PET molecules, GL-g-LMPET nanofillers were homogeneously dispersed in PET matrix. Thermal properties and crystallization properties of obtained nanocomposites were systematically characterized using Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD), and Thermo Gravimetric Analysis (TGA). Results show that GL-g-LMPET nanofillers could improve the thermal stability of PET, e.g., increase up to 16.6 °C in temperature at the maximum rate of weight loss. In addition, the GL-g-LMPET also acts as an efficient nucleating agent for PET, exhibiting (1) higher crystallization temperatures; (2) higher degrees of crystallinity; and (3) faster rates of crystallization.In this article, graphene oxide nanosheets grafted with low molecular weight poly were in situ synthesized via carboxylation, acyl chlorination and grafting modification in order to improve the compatibility between GO and PET phases and enhance the thermal stability and crystallization properties of PET. Fourier Transform Infrared (FTIR), X-ray Photoelectron Spectroscopy (XPS), and Atomic Force Microscopy (AFM) characterization results demonstrated that LMPET chains have been successfully grafted onto the surface of GO. To further investigate the influence of modified GO on properties of PET, modified PET was prepared by incorporating the GL- Among all the nanoparticles, graphene oxide (GO) has attracted considerable research attention due to its excellent thermal stability , electrig-LMPET nanocomposites. Due to the fact that the polar parameter of LMPET grafted on the surface of GO was similar to that of PET matrix, it is beneficial to increase the interfacial compatibility between PET and GO phases, and eventually improve thermal stability and crystallization properties of PET. The structure of GL-g-LMPET as well as the thermal property and crystallization behavior of its composites were correspondingly discussed to provide comprehensive knowledge on the influence of modified GO on the PET matrix, providing fundamentals for the preparation of other high-performance composites using functionalized GO. In this work, to improve dispersion and interfacial strength, functionalized GO nanosheets were synthesized via a three-step reaction through a new approach. GO was pre-modified with carboxylation and acylation and then grafted with low molecular weight poly (LMPET) via in situ polymerization. Carboxylation reaction was used to provide more reactive sites and acylation reaction to obtain higher reactivity, thus achieving a complete surface modification of GO. The surface-functionalized GO nanosheets were further added into the PET matrix by melting blending to obtain the PET/GL-4 with a intrinsic viscosity of 0.676 dL/g). Other reagents used in this work were provided by Sinopharm chemical Reagent Co., Ltd.  All of the regents were used as received without further purification.Natural flake graphite was purchased from Qingdao Daewoo Graphite Co., Ltd. . PET was supplied by Sinopec Yizheng Chemical Fiber Research Institute, Yizheng, China, : 20.0 mg graphene oxide (>90% purity) prepared using Hummers method , 1.2 g sSecondly, synthesis of acyl chloride-functionalized graphene oxide (GL): 20.0 mg GH was added into benzene and treated under ultrasonic for 2 h. Then, 10 mL thionyl chloride was added under the protection of nitrogen flow. The mixture was heated to 65 °C and reacted for 6 h. Finally, the resultant solution was washed using benzene to remove the non-reactive thionyl chloride in a high-speed centrifuge. Based on the original feed ratio of GO nanofiller and thionyl chloride matrix, the resulting GL mass fraction was determined to be 9.4 wt %.g-LMPET: 11.2 g DMT , 7 mL EG (ethylene glycol) and a small amount of zinc acetate were mixed in a three-necked flask and reacted under 190 °C for 2 h. After that, trace amounts of Sb2O3 used as catalyst and 0.5–1 drops of triphenyl phosphate acted as thermal stabilizer were added into the mixture, and further stirred for 1 h followed by the addition of 10 mg GL. The mixture was then heated to 230 °C and allowed to remain for 2 h. The obtained mixture was centrifuged and washed using phenol/carbon tetrachloride (m:m = 1:1) to remove ungrafted LMPET. Finally, the product was dried under vacuum at 80 °C for 24 h. Additionally, graphene oxide—LMPET (GO-g-LMPET) was prepared using a similar method  under 285 °C. The in situ modified graphene oxide nanosheets and PET were fully melt-blended, and the content of GL-g-LMPET nanosheets in the matrix was 0.5 wt %. For comparison, pure PET, PET/GO and PET/GO-g-LMPET nanocomposites were prepared according to the above procedure described for PET/GL-g-LMPET nanocomposites.PET/GL-−1. X-ray photoelectron spectra (XPS) were obtained from an ESCALAB250Xi-XL electron spectrometer  at a using 150 W Al Kα radiation. A surface microstructure of GO nanoparticles was observed in a high resolution transmission electronic microscope . Atomic force microscopy  was used to determine the thickness of GO and modified GO at ambient temperature. Surface morphology analysis of PET and its nanocomposites were carried out by means of a scanning electron microscope . Contact angle measurement (CAM) and surface free energy estimation of the nanocomposites were carried out at room temperature on a JY-82 contact angle system . The average contact angle from six different locations on each nanocomposite was determined and the experimental uncertainty was within ±2°. X-ray diffractometer  was used to test the crystalline properties of PET/GL-g-LMPET nanocomposites. The scanning was ranged from 5° to 60° with scanning speed of 2 (°)/min. DSC  was used to characterize the thermal properties of PET based nanocomposites. Initially, samples were heated to 300 °C with a ramp rate of 10 °C/min and kept for 5 min to remove the thermal history. Then, samples were cooled and re-heated from 25 to 300 °C with a ramp rate of 5/10/15/20 °C/min. The crystallization and melting data were obtained from second cooling and heating curves. Thermogravimetric analysis (TGA) was performed on a PerkinElmer Diamond thermal analyzer . Samples were heated from 50 to 600 °C nitrogen with a ramp rate of 10 °C /min under a continuous nitrogen flow rate of 20 mL/min. Fourier transform infrared (FTIR) spectra were recorded on a Nicolet 5700 FTIR spectrometer  over the range of 4000–500 cmCX) for the pure PET and nanocomposites were determined from the DSC traces by the enthalpy variation during the second melting scan using the following Formula (1): mH is the enthalpy of melting of the measured sample, m is the quantity of GL-g-LMPET, and ΔH0 is the enthalpy of melting of 100% crystalline PET, reported to be 140 J/g [The degree of crystallinity ( 140 J/g . tX) of PET and its composites at different times calculated according to Formula (2):dHC/dt is the heat flow rate, t0 and t∞ are the time at which crystallization starts and ends, respectively.The relative crystallinity (g-LMPET (0.5 mg/mL) in water and Phenol/C2H2Cl4 solution after 3 min of the sonication process were presented in 2O and Phenol/C2H2Cl4, a clear interface was observed (as marked with a red line). Since a large number of hydrophilic groups exist on the surface of GO, GO showed a homogeneous dispersity in aqueous phase (as shown in 2H2Cl4) [2O to Phenol/C2H2Cl4 after chemical modification. This phenomenon could be an indicator to some extent of the successful grafting of LMPET chains onto the surface of GO nanosheets [The dispersion of GO and GL- 2H2Cl4) . Therefonosheets . −1 (O–H stretching vibration), 1409 cm−1 (O–H deformation vibration) and 1052 cm−1 (epoxide groups stretching vibration) [−1, suggesting the successful reaction between COOH groups and acyl chloride. Based on the data, GL-g-LMPET was further synthesized and the FTIR spectrum was shown in −1 corresponding to C–O–C and C=O vibrations of PET [−1 indicates the C–H bond vibration of the benzene ring [g-LMPET, XPS was used to measure the near-surface composition and examine the valence states of the observed elements. C1s spectra of GO and GL-g-LMPET are shown in To further confirm the structure of modified GO, FTIR was applied to characterize the structure of GO and modified GO, as shown in bration) ,27 decres of PET ,33 appeag-LMPET. g-LMPET is entirely different as shown in g-LMPET sheets can be also observed in g-LMPET monolayers [g-LMPET were 1.09 and 1.65 nm, respectively. Thus, it has also been confirmed that the functionalization process has changed the morphology of GO sheets.TEM was utilized to investigate the microstructure of GO and GL-nolayers . As seeng-LMPET can reflect the grafted LMPET molecular chains content to a certain degree due to the fact that the synthesized nanosheets were Soxhlet extracted for 72 h to remove unreacted polymers. For the TGA curve of GO, the initial mass loss appeared at around 100 °C, mainly attributing to the evaporation of water molecules adsorbed on the surface of GO. The major weight loss of GO appeared at 150~200 °C, which was caused by the pyrolysis of GO sheets [g-LMPET and GL-g-LMPET are completed at 270~370 °C and 350~430 °C, respectively, which occurs later than that of the GO, illustrating that the grafting LMPET is effective for enhancing the thermal stability of GO [g-LMPET (40 wt %) and GL-g-LMPET (27 wt %) under 700 °C, the weight percent of grafted LMPET onto GL and GO nanosheets were roughly calculated as 18 and 5 wt %. The comparatively higher grafting content of GL-g-LMPET may be benefited from the high active precursors (GL with multiple reactive sites).The grafting content of LMPET was characterized using TGA, as shown in O sheets . Howeverty of GO . Moreoveg-LMPET nanocomposites in g-LMPET sheets can be well dispersed in the matrix, as no large agglomerates can be found. This demonstrates that the interface between GL-g-LMPET sheets and the PET matrix is stronger than that in the PET/GO and PET/GO-g-LMPET nanocomposites.It is well known that the good dispersion of nanoparticles and the strong interfacial interaction are two important factors to improve the physicochemical properties of nanocomposites modified polymer. SEM was applied to investigate the dispersion and interface of various nano-GO into the polymer matrix. D) surface free energies of various nanocomposites, which are presented in g-LMPET and PET/GL-g-LMPET nanocomposites. In contrast to PET/GO and PET/GO-g-LMPET nanocomposites, the GL-g-LMPET particles embedded in the PET matrix significantly decreased the average contact angle of the probe liquids. This shows that the functionalized GO nanoparticles can increase γD of the PET nanocomposite. In other words, unfunctionalized GO and imperfect functionalized GO-g-LMPET have a strong tendency to agglomerate because of high surface free energy, resulting in less level dispersion in the PET matrix. However, the surface-functionalized GO nanoparticles process similar polarity with PET, which is beneficial to increase the interfacial compatibility and alter the orientation of the PET chains at the surface of nanocomposite, thus resulting in an increase in the γD of the resulting nanocomposites. In short, surface-functionalized GO nanoparticles tend to reside in the polymer body [We further used contact angle measurement to characterize the dispersion of nanoparticles on the surface of nanocomposites. Herein, we applied water probe solutions to study the dispersed  of PET/GL-g-LMPET nanocomposites notably increased as compared with those of pure PET, PET/GO and PET/GO-g-LMPET, whereas the PET/GO and PET/GO-g-LMPET only performed trivial changes. g-LMPET was added into the PET matrix. The lower melting temperatures of PET nanocomposites were similar with that of PET, suggesting that the lower Tm was caused by the crystals of PET. For the higher melting temperature (Tm), the perfection degree of crystals was relatively high, which may be caused by the crystals of PET/ GL-g-LMPET. Moreover, SEM of GL-g-LMPET can further clarify the two different kinds of PET crystals in nanocomposites (g-LMPETs were dispersed homogeneously in the PET matrix (as pointed at by white arrows), demonstrating a strong interaction between GO nanosheets and PET matrix. Thus, two different regions appeared in the PET matrix, namely GO surrounded by PET molecular and PET without GO. The former region formed crystals with higher perfection due to the strong interaction between GO and PET moleculars compared with that of the PET region, leading to the appearance of two different melting temperatures. The effect of different nucleating agents on thermal behavior of PET matrix during heating and cooling processes was characterized by DSC ,42, as smposites d. GL-g-Lg-LMPET and GL-g-LMPET on the crystalline structure of PE in g-LMPET and PET/GL-g-LMPET, corresponding to the (011), (010), (110) and (100) diffraction planes, respectively [g-LMPET and GL-g-LMPET. It indicates that GL-g-LMPET acts as a kind of more high-effect nucleating agent to improve the crystallization ability of PET. To further investigate the crystallinity of PET and PET nanocomposites, the relative crystallinity XC was calculated by Equation (2) and listed in g-LMPET nanocomposite exhibits the optimal crystallization behavior and its crystalline increased by 6% more than pure PET. The XRD patterns provide important insight into the effects of GO, GO-ectively . This isg-LMPET and GL-g-LMPET. Based on the above DSC crystallization curves and Formula (2), non-isothermal crystallization process of PET and its composites at different cooling rates were obtained and shown in 1/2, to characterize the crystallization rate , and the lower t1/2 values indicate higher crystallization rate. Obviously, at the same cooling rate, the t1/2 values decreased in the order of PET/GO, PET/GO-g-LMPET and PET/GL-g-LMPET. PET/GL-g-LMPET achieving the higher crystallization rate is attributed to the good dispersion and strong interfacial adhesion of GL-g-LMPET in PET.Another criterion commonly used to evaluate crystallization property is the crystallization rate of nanocomposites. DSC of all the samples at different cooling rates were performed and relevant results are presented in g-LMPET samples were at higher temperatures . The Tmax of PET/GO, PET/GO-g-LMPET and PET/GL-g-LMPET nanocomposites were increased by approximately 5.3, 7.4 and 16.6 °C, respectively , and the crystallization temperature was also increased by 9 °C along with the crystallinity (XC) increment up to 29.7%. Moreover, the crystallization rate (Xt) of PET/GL-g-LMPET nanocomposites was simultaneously enhanced. This work represents a novel and effective functionalization strategy to improve the dispersion and interface interaction of GO in a polymeric matrix and brings significantly enhanced properties, therefore facilitating industrial applications of PET in a wider range.In this research, a novel strategy to significantly enhance thermal stability and crystallization properties of PET was demonstrated. Carboxylation and acyl chlorination provide more reactive points and promote the grafting reaction of LMPET, achieving a complete surface modification of GO. In addition, the similar polarity of LMPET and PET contribute to improving the dispersion and interface interaction of GO in a polymeric matrix. Due to the addition of GL-


Temporal-envelope cues are essential for successful speech perception. We asked here whether training on stimuli containing temporal-envelope cues without speech content can improve the perception of spectrally-degraded (vocoded) speech in which the temporal-envelope  is mainly preserved. Two groups of listeners were trained on different amplitude-modulation (AM) based tasks, either AM detection or AM-rate discrimination , while an additional control group did not undertake any training. Consonant identification in vocoded vowel-consonant-vowel stimuli was tested before and after training on the AM tasks . Following training, only the trained groups showed a significant improvement in the perception of vocoded speech, but the improvement did not significantly differ from that observed for controls. Thus, we do not find convincing evidence that this amount of training with temporal-envelope cues without speech content provide significant benefit for vocoded speech intelligibility. Alternative training regimens using vocoded speech along the linguistic hierarchy should be explored. Temporal-envelope information is crucial for speech perception –10. For Intelligibility of vocoded speech for listeners with normal hearing depends on several factors, but most importantly the number of frequency bands used for vocoding , 21. WhePerceptual learning of TE information without speech content  has been observed after only two days of training . GeneralOne of the pioneer investigations exploring the relationship between training on low-level tasks and performance on different language outcomes was the study by Merzenich and colleagues . They shGiven the importance of TE cues to both aided and unaided speech perception, we asked here whether (1) listeners’ ability to perceive these envelope cues in non-speech stimuli is related to initial learning of vocoded speech, and (2) training on these cues can transfer to improved perception of spectrally degraded speech. To investigate this, we trained listeners on either detection of TE modulation  or the discrimination of the AM rates, and tested for improvement in perception of vocoded vowel-consonant-vowel (VCV) ‘nonsense’ syllables. These sub-lexical stimuli were chosen to minimize the influence of higher-level linguistic structure  on learning effects. We predicted that (1) listeners’ performance on AM tasks prior to training will be correlated with their ability to quickly adapt to vocoded VCV segments on initial exposure (pre-training), and (2) training on TE cues without speech content would improve vocoded consonant identification when compared to untrained, control listeners.The research protocol was approved by the Nottingham University Hospitals NHS Trust Research Ethics Committee. Written informed consent was obtained from all participants prior to the first experimental session, and they were paid an inconvenience allowance for their participation.Fifty-four native English speakers  aged 18–40 years  were recruited via posters from the University of Nottingham student population and the general public. No participants reported any history of language disorders or hearing impairment. Screening confirmed that bilateral audiometric pure-tone thresholds were ≤ 20 dB Hearing Level at octave frequencies across 0.25–4 kHz in both ears. No participants had prior experience of psychoacoustic testing or vocoded speech.Testing took place within a double-walled sound-attenuating booth. Tasks were based on custom software written in MATLAB Release R2007a . Stimuli were presented diotically at 70 dB sound pressure level (SPL) using Sennheiser HD-25-I headphones. Participants responded to vocoded speech recognition tasks via a touchscreen LCD computer monitor placed in front of them at a comfortable distance. Responses to psychophysical tasks were recorded via a custom-made button box placed horizontally in front of the listeners.The study was conducted over four consecutive days and the protocol consisted of a pre-training phase, a pre-test, a training phase, and a post-test . During Since previous studies have shown a relationship between listeners’ memory span and performance on AM tasks, as well as the perception of vocoded speech e.g., , 34), al, al34]),All participants undertook pre-training on the vocoded consonant identification task. This phase allowed us to match the groups on initial average speech identification performance by assigning participants to each group according to their baseline performance and thus avoid bias due to poorer listeners showing greater learning  were represented orthographically on a touch-screen and participants were instructed to select the response alternative corresponding to the consonant they heard. There was no time limit for entering the responses and participants were encouraged to guess if they could not identify the consonant. After each response, auditory and visual feedback were given in the form of the clear unprocessed version of the stimulus, followed by a repeat presentation of the vocoded VCV and the correct consonant highlighted in green on the screen. The presentation of the stimuli was fully randomized for each participant and block. To familiarize participants with the task, five practice trials were presented before the first block.www.tigerspeech.com, Version 1.05.02). Speech stimuli were divided into six contiguous frequency bands equally spaced on the ERBN-number scale. Corner frequencies (3-dB down from the peak of the passband) of the filters were at 100, 331, 725, 1396, 2538, 4485 and 7800 Hz with a roll-off of 24 dB per octave. The frequencies of the 6 channels were centred at 185, 491, 1007, 1881, 3375, and 5916 Hz. The slow-varying envelope within each band was extracted by removing pitch-synchronous oscillations above 45 Hz (with a roll-off of 24 dB per octave) and used to modulate sine waves located at the logarithmic center frequency of each analysis band. Although classic psychophysical studies 0.5 (where m is the modulation depth) to cancel out the increase in power resulting from amplitude modulation, and thus avoid participants’ decisions based on intensity cues [AM stimuli were created by multiplying a 1-kHz pure-tone with DC-shifted sinusoids of the required modulation rate. For the pre- and post-tests, a modulation rate of 8 Hz was used to maximize the potential transfer of learning to vocoded consonant identification, as this corresponds to temporal information at the sub-syllabic level .ity cues . The stam) was initially set to 100% (0 dB), then adaptively adjusted by a factor of 1.58 (4 dB) according to a one-down, one-up staircase procedure. After the first incorrect response, the factor was changed to 1.26 (2 dB) and an adaptive three-down, one-up staircase procedure, targeting 79% correct on the psychometric function [m = 100%), and two ‘impossible’ (m = 0%).In the AMD task the modulation depth (function  was usedm = 100%) and the target interval contained the same carrier modulated at a higher rate. The modulation rate (fm) for the target varied adaptively (8 Hz + Δfm), starting at Δfm = 50% of the standard rate  and was reduced by a factor of 1.4, according to a one-down, one-up staircase procedure. After the first incorrect response, the step size was changed to √1.4 and a three-down, one-up staircase procedure. Five practice trials were presented before the first block to familiarize participants with the task. Three of these trials were ‘easy’ (Δfm = 50%) and two were ‘impossible’ (Δfm = 0%).The procedure and task for AMRD was similar to those used for AMD. Two blocks of 30 trials were presented to each listener employing a 3I-3AFC oddball design with feedback. The two standard intervals consisted of 1-kHz carriers modulated at 8 Hz (f). Listeners were instructed to indicate the interval that was different in pitch. Starting with Δf = 50% , Δf of the subsequent trial was divided by two until the first incorrect response. Thereafter, the step size was √2. Before the first block a five-trial demonstration was administered. Three of these were ‘easy’ (Δf = 50%) and two impossible (Δf = 0%). FD stimuli consisted of 100-ms tones (including 10-ms raised cosine ramps) with an inter-stimulus interval of 500 ms. This task was chosen as a control psychophysiological task in order to show the degree of specificity of the AM training regimes, and to be able to measure any disruption of learning due to training on AMD or AMRD tasks.Two blocks of 30 trials were administered to every listener employing the same 3I-3AFC oddball design with feedback. The frequency of the two standard tones was 800 Hz while the frequency of the target, randomly determined interval contained a higher-frequency  occur–speech ranges that are critical for performing the speech perception task (vocoded consonant identification). In English the syllabic rate is around ~4 Hz [One group was trained on an AMD task (‘AMD-trained’ group) and a second group was trained an AMRD task (‘AMR-trained’ group), using similar adaptive procedures to those used for the test phases. Participants completed 21 blocks of 60 trials spread across two days. Nine blocks were completed on Day 2 (immediately following the pre-test) and an additional 12 blocks were completed on Day 3. For both AM training tasks, the standard nd ~4 Hz , 41, thend ~4 Hz ), and thnd ~4 Hz , 44). Thlme4 [lme). Significance p-values and Type III F-statistics for main effects and interactions were calculated using Satterthwaite approximations to denominator degrees of freedom as implemented in the lmerTest package [2-statistics for main effects and interactions were calculated using the car package [Analyses were conducted using the lme4  package lme4 . Continu package . For the package . Type II package . We alsoAll models included group  and test  as fixed effects, including all main effects and their interactions. Additionally, information transfer analyses also included type of phonetic feature  as a fixed effect, including all main effects, 2-way and 3-way interactions. All models included maximal within-unit random effects structure , 53, thu10-transformed and fitted with a psychometric function using the Psignifit toolbox [rd and 1st quartile from each group and task were considered outliers and excluded from further analysis (1.9%). On average the number of reversals per block of 30 trials (pre- to post-test phase) in each task was: AMD = 5.53 (SD = 1.72); AMRD = 5.11 (SD = 1.63); FD = 5.70 (SD = 2.16). Threshold estimates for the two consecutive blocks of 30 trials in pre- and post-tests were highly correlated: AMD pre-test: r = .75, p < .001, AMD post-test: r = .62, p < .001; AMRD pre-test: r = .70, p < .001, AMRD post-test: r = .53, p < .001; FD pre-test: r = .74, p < .001, FD post-test: r = .89, p < .001.Raw threshold data from psychophysical measures  were log toolbox . Tracks The threshold estimates for each task were analyzed using a group  × test (pre-test and post-test) mixed-effects model. Since all participants conducted two blocks of each task in the pre- and post-test sessions, this factor  was included as a control variable in the fixed-effect structure of the model see .2 (2) = 1.93, p = .38). It is worth noting that pre-test intercepts were not statistically different between trained groups and the control group  = 23.41–69.51; AMD-trained: 58.9%, SD = 49.2, CI (95%) = 22.0–70.9; AMR-trained: 63.3%, SD = 48.2, CI (95%) = 22.7–70.2; see  .13 see —Table N, .13 see .rd and below the 1st quartile of the group were considered outliers and discarded (3.7%). Due to an experimenter error one participant performed the consonant identification task with feedback in the post-test, and therefore was excluded from further analyses  analysis  was condContrary to our prediction and the findings from Erb and colleagues , 18, prePrior to training the groups did not differ statistically on AMD  = .63, p = .53), AMRD  or FD  = .69, p = .51), nor the summary-based statistics showed reliable differences across groups see . LearninOnly the AMD-trained group showed reliable improvement on the AMD task  = 1.39, p = .50). It is worth noting the large and overlapping confidence intervals for the differences in improvement from pre- to post-test in each group, with lower confidence intervals close to 0 values even for AM-trained groups. As can be also seen in 2 (1) = 12.5, p < .001; see AM-trained groups, but not controls, showed a significant improvement on vocoded consonant identification between pre- and post-test  = 492.6, p < .001), with manner best received (77.5%), followed by voice (73.9%) and place of articulation (32.5%), consistent with results reported by Lorenzi et al. (2000). Pre- to post-test learning was also significant  learning observed in the untrained controls. Furthermore, we failed to find significant correlations between listeners’ initial learning on vocoded VCVs and their performance on AM tasks. It is worth noting that the sample size of this study as well as the studies from which the initial hypothesis are drawn are small. This might lead to inconsistent results across studies given the binary interpretation of the results (significant versus non-significant effects). Here we have reported summary-based descriptive statistics alongside the more traditional inferential statistics as per to show the level of uncertainty associated with our results. As can be observed in the results section see also  confidenWe specifically chose the vocoded consonant identification task as an outcome measure because, lacking lexico-semantic cues, it presumably relies more heavily on bottom-up, perceptual processing compared with more complex speech materials such as words or sentences. We reasoned that the effects arising from training on TE cues would be strongest for a task that relies on low-level processing. However, the choice of speech material and the experimental procedure may also have hindered our ability to attain observable transfer of learning. Given the closed set of stimuli, the benefits of longer term AM training might be dwarfed by the rapid learning that occurs via repeated exposure to the stimuli (see ). This lAlthough both of these explanations are plausible, a third alternative should be considered: that the mechanisms involved in learning to distinguish of AM tones are not the same as those involved in learning of vocoded VCVs. This interpretation is supported by the lack of correlation between initial thresholds on AM tasks and learning of vocoded VCV during pre-training. Furthermore, correlation analysis between the amount of learning for each trained group and the amount of generalization to VCV perception also fail to show a significant relationship . Hence, it could be hypothesized that whilst perception of TE information relies on detection and discrimination of low-level acoustic features, perceptual learning of vocoded speech involves adjusting the mapping of low-level acoustic feature cues present in the degraded speech signal onto pre-existent phonological representations e.g., , 65, 66), 6665, 6We found no transfer of learning between the AMD and AMRD tasks even though they used very similar stimuli and identical testing paradigms. Maidment et al.  also shoA final possibility is that transfer was not achieved because the AM training was limited to modulations frequencies ≤ 16 Hz and a single carrier frequency, whereas the VCV stimuli contained modulation information up to ~45 Hz along several carrier frequencies (or bands). Since AM learning can be both carrier-  and moduOur findings cast doubts on the role of low-level perceptual learning in tone-vocoded consonant perception beyond initial and rapid learning. We showed that the ability of naïve listeners to process TE cues was not related to the improvement in their ability to perceive vocoded nonsense syllables, and that although AM training as used here improved perception of VCV speech material, improvement did not substantially differ from that observed for the untrained control group. Hence, improvements did not appear to be directly related to TE information training in our sample of participants, but rather to repeated exposure to the specific speech stimuli (see ). We theS1 AppendixDetailed information of the statistical models employed, including tables with results and significance values.(PDF)Click here for additional data file.S2 AppendixDetailed information of the consonants used in the experiment as a function of manner, voice and place of articulation and the procedure to compute IT scores, including script written in R.(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S1 Dataset(TXT)Click here for additional data file.S2 DatasetParticipants’ scores for all blocks of vocoded consonant identification task, including hits and misses for each specific phonetic feature.(TXT)Click here for additional data file.S1 TableMeans, standard deviations, and 95% confidence intervals fore each group and session.(PDF)Click here for additional data file.S2 TableCorrelations between hearing and cognitive tasks before undertaking any training.(PDF)Click here for additional data file.S1 Audio FileFile containing vocoded /ama/ non-sense syllable.(WAV)Click here for additional data file.S2 Audio FileFile containing vocoded /isi/ non-sense syllable.(WAV)Click here for additional data file.S3 Audio FileFile containing vocoded /ugu/ non-sense syllable.(WAV)Click here for additional data file.S4 Audio FileFile containing clear /ama/ non-sense syllable.(WAV)Click here for additional data file.S5 Audio FileFile containing clear /isi/ non-sense syllable.(WAV)Click here for additional data file.S6 Audio FileFile containing clear /ugu/ non-sense syllable.(WAV)Click here for additional data file.S1 FigMeans, standard deviations, and 95% confidence intervals fore each group and session.(PDF)Click here for additional data file.


MMP-8 and GSS mRNA levels were increased in 12 samples in patients with pyelonephritis 3.27 (p < 0.01) and 1.94 (p < 0.001) times, respectively. We found a strong and significant correlation (p < 0.001) between the investigated mRNA for IL-6 and MMP-8, IL-6 and GSS, MMP-8 and GSS. Moderate degree of correlation was established between IL-6 and the typical para-clinical indicator of leucocytes  and between GSS and leucocytes . Salivary IL-6, MMP-8 and GSS mRNA levels in combination with urine test analysis could be useful diagnostic tool for the very distributed disorder of pyelonephritis in childhood.Nowadays, saliva is a subject of growing scientific interest because of its definite advantages as diagnostic medium. The aim of our study was to investigate the diagnostic potential and reliability of messenger RNAs (mRNAs) of selected genes—interleukin-6 (IL-6), matrix metalloproteinase-8 (MMP-8) and glutathione synthetase (GSS)—as salivary markers in children with diagnosed pyelonephritis and to correlate their levels with typical urine para-clinical indicators of the disease. Analysis of the mRNA levels for IL-6, MMP-8 and GSS in 28 children hospitalized with the diagnosis of pyelonephritis was conducted applying the method of quantitative reverse transcription polymerase chain reaction (RT-qPCR). In the study group ( Nowadays there is a constant increase of the scientific interest in the application of minimally invasive diagnostic procedures . Saliva Urinary tract infections (UTIs) are characterized by high prevalence in different periods of childhood, especially during the stages of early childhood and preschool age . These dDifferent molecules are involved in the inflammatory process and some of them could serve as reliable biomarkers for the state and progression of specific inflammatory diseases. It is known that the pro-inflammatory cytokine IL-6 is implicated in renal inflammatory diseases. Specific cells of kidney tissue, respectively tubular epithelial cells, mesangial cells, endothelial cells and podocytes, have the potential to secrete IL-6 . Matrix The investigated salivary biomarkers—IL-6, MMP-8 and GSS—re directly involved in inflammatory process. Scientific literature confirms that they increase in conditions of pyelonephritis—an inflammatory process of the excretory system ,46,47,48p < 0.001)  .n = 12) of all the investigated children (p < 0.01) (n = 16) showed a statistically non-significant 2.66-fold decrease of the expression (p < 0.001). This indicates a strong correlation between both indices.The investigated group of 28 children with pyelonephritis could be split into two subgroups, based on the results obtained for the levels of the three investigated salivary parameters. For MMP-8, we have established a statistically significant 3.27-fold increase of the gene expression only among 42.86% ( < 0.01) . The othpression . Pearsonn = 12) of all of the investigated children (p < 0.001) (n = 16) marked by reduction of the GSS expression were without any statistical significance (p = 0.062) (p < 0.001) between GSS and IL-6, and also a strong correlation between GSS and MMP-8 equaling to 0.86 (p < 0.001). A 1.94-fold increase of GSS expression with statistical significance was established for 41.38% (< 0.001) . The oth= 0.062) . Almost = 0.062)  also sho= 0.062)  and vice= 0.062) . VariatiAmong all 28 participants with diagnosed pyelonephritis, nine showed considerably high mRNA levels of IL-6, MMP-8 and GSS . Six of these nine patients showed statistically significant results for the three investigated parameters  . Two of Concerning both patient 12 and 28, we obtained not only the highest mRNA levels of all the investigated salivary biomarkers, but we also established the greatest values of para-clinical indicators—leucocytes and blood in urine analyses—compared to all the participants , Table 1p < 0.05) and between GSS and leucocytes  (p = 0.4459) and between GSS and blood . Simultaneously, a similar slight degree of correlation without statistical significance was registered between the salivary MMP-8 and proteins in urine samples, amounting to 0.16 (p = 0.4236)  . These r 0.4236) .Saliva has a lot of advantages as a biological fluid in the laboratory diagnostics, especially among children. It is easy to collect and rich in different analytes, including protein, DNA and RNA originating from different body parts . Care shIn the present study, we have examined the mRNA levels of IL-6, MMP-8 and GSS using saliva as an emerging, non-invasive and perspective medium for precise diagnosis of acute pyelonephritis in children. Based on the investigation of the mRNA levels of these three parameters, we recorded that the level of IL-6 was considerably higher compared to MMP-8 and GSS . This reOn the other hand, it was expected that the highest levels of IL-6, MMP-8 and GSS were established among two of the patients, 12 and 28 . AccordiWe established a strong correlation between the salivary indicators IL-6 and MMP-8. These parameters are associated with the already confirmed functions of MMP-8 in conditions of acute inflammation , includiWe established a direct relationship between the elevated mRNA levels of IL-6, MMP-8 and GSS among six of the patients, namely patients 12, 16, 17, 19, 23, 28 . This inThe established moderate degree of correlation between IL-6 and leucocytes, moderate degree of correlation between GSS and leucocytes, slight degree of correlation between IL-6 and blood and GSS and blood, as well as between MMP-8 and proteins are principally associated with the pathological alteration and pathophysiological traits of pyelonephritis. Compared to chronic renal failure, the excretory system disorder pyelonephritis is not accompanied by the major impact of MMP-8 upon epithelial architectonics and function. The low rate of impingement of the epithelial tissue in the state of pyelonephritis corresponds to the obtained results illustrating a correlation between blood in urine on one hand, and IL-6 and GSS in saliva, on the other . In the Both the established significant correlation between GSS and IL-6 and the strong correlation between GSS and MMP-8 are related to the fact that oxidative stress is also influenced in inflammatory processes ,63,64. RPresent data concern a pilot study with an accent on the potentials of saliva as a diagnostic tool. Further investigations with definite tendency of increase of the number of participants suffering from the common health disorder of pyelonephritis will contribute to proper evaluation of mRNA levels of IL-6, MMP-8 and GSS as salivary biomarkers. An informed consent was given and a declaration of informed consent was signed by parents or legal guardians of children or another person accompanying each child participating in the research. The University Medical Ethical Institutional Board of the Medical University of Varna approved the use of the collected salivary samples from children with diagnosed pyelonephritis and healthy controls for the purposes of our study.Participants between 0–18 years of age.Children with established pyelonephritis.A control group of children without any common health disorders.Inclusion criteria:Patients of pyelonephritis older than 18 of age.Patients of clinical manifestation of periodontal diseases, respectively children with loss of bone tissue, loss of clinical attachment level or formation of periodontal pockets.At the time of sample collection, patients do not suffer from other inflammatory diseases.Do not suffer from autoimmune disorders or malignant diseases.No anamnestic data of allergic reactions’ backgroundDo not use immunosuppressive drugs, no corticosteroids’ application, no antihistamins’ medication.Exclusion criteria:Therapy of all of these patients includes antiobiotics of the groups of penicillins, cephalosporines, macrolides, as well as non-steroid anti-inflammatory drugs.Our study included 28 children suffering from pyelonephritis. A control group of nine children with no common health disorders was also included in the investigation. The patients were hospitalized at the Department of Pediatrics of St. Marina University Hospital. The gender distribution was as follows: 17 of the patients were female and 11 were male. A total number of four male and five female healthy children represented the control group. The healthy participants were outpatients of the University Dental Medicine Center at the Faculty of Dental Medicine, Medical University-Varna. Anamnestic data of each patient was obtained from their individual medical history.The procedure of saliva sample collection was performed at the bed of the patient individually. Healthy children were asked to provide saliva in outpatient conditions at the dental office. The samples of unstimulated whole saliva were collected in sterile DNase- and RNase-free collection tubes, frozen immediately on dry ice, and stored at −80 °C until further analysis. The saliva specimen collection was carried out in the time interval between 9.00 a.m. and 11.30 a.m. All the participants in the investigation were instructed to properly brush their teeth just before the sample collection. Frozen saliva was stored for a period of approximately 18 months.For the planned urine assays are taken morning urine samples. There is a requirement to be provided second portion maximum clear urine samples. A small amount of urine had to be collected into individual sterile container. Immediately after urine collection, the container was tightly closed. ® 2xGreenStarTM qPCR Master Mix . Reactions were performed in 96 well plates under the following conditions: predenaturation and enzyme activation 95 °C/10 min, denaturation 95 °C/30 s, annealing 60 °C/30 s, extension 72 °C/30 s, 45 cycles. Each gene was analyzed in triplicate for all samples. Ct values were detected using ABI PRISM 7500 software . Gene expression levels were calculated using the 2−∆∆Ct method [Total RNA extraction was conducted using 200 µL unstimulated whole saliva and the TRIzol RNA extraction method . Evaluation of RNA concentration and purity was performed spectrophotometrically using Synergy 2 Multi-Detection Microplate Reader . Before conducting RT-PCR DNase, treatment of total RNA was performed using DNase I recombinant kit  following the manufacturer’s instructions. For reverse transcription reaction 100 ng DNase-treated total RNA was added and cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit  with oligo (dT)18 primer. Primers for real-time PCR were commercially synthesized . Primer sequences were as follows: human actin beta forward 5′-CTGGAACGGTGAAGGTGACA-3′, reverse 5′-AAGGGACTTCCTGTAACAATGCA-3′; human IL-6 forward 5′-GGCACTGGCAGAAAACAACC-3′, reverse 5′-GCAAGTCTCCTCATTGAATCC-3′; human MMP-8 forward 5′-GCAACCCTATCCAACCTACTG-3′, reverse 5′-CATCCTCAGCTACAAAGAGTCG-3′; human GSS forward 5′-TACGGCTCACCCAATGCTC-3′, reverse 5′-GCACGCTGGTCAAATATGTTTC-3′. SYBR Green qPCR analysis was performed using AccuPowert method  and exprFor the hospitalized participants into the study has been implemented the routine chemical method of identification of proteins in urine samples. By means of a quantitative microscope, urine test were evaluated the levels of red blood cells and white blood cells. The microscope tests were performed after centrifugation of urine samples and the obtained sediment was used to examine the presence of white and/or red blood cells. p-values < 0.05 were considered statistically significant. We used a correlation analysis with calculation of Pearson’s coefficient. The latter assesses the significance of linear interrelation between two variables. The values of Pearson’s coefficient in the range between 0.00 and 0.25 correspond to slight correlation. The values in the range from 0.26 to 0.49 are associated with moderate correlation. Levels of the coefficient from 0.50 to 0.69 are related to considerable correlation. The values from 0.70 to 0.89 characterize strong correlation. The values from 0.90 to 1.00 are characteristic of a very strong correlation. Statistical analysis t-test was performed using Microsoft Excel Office 2007 software, Salivary IL-6, MMP-8 and GSS mRNA levels in combination with urine test analysis could be a useful diagnostic tool for the very distributed disorder of pyelonephritis in childhood.


We present results of a multicenter study using an updated version of UBC®Rapid Test in bladder cancer patients, patients with urinary bladder cancer positive history, and healthy controls. Material and Methods: In total 530 urine samples have been included in this study. Clinical urine samples were used from 242 patients with tumors of the urinary bladder (134 non-muscle-invasive low-grade tumors (NMI-LG), 48 non-muscle-invasive high-grade tumors (NMI-HG), and 60 muscle-invasive high-grade tumors (MI-HG)), 62 patients with non-evidence of disease (NED), and 226 healthy controls. Urine samples were analyzed by the UBC® Rapid point-of-care (POC) assay and evaluated by Concile Omega 100 POC Reader. All statistical analyses have been performed using R version 3.2.3. Results: Elevated levels of UBC® Rapid Test in urine are higher in patients with bladder cancer in comparison to the control group (p < 0.001). The sensitivity for the whole bladder cancer cohort was 53.3%  90.2%, negative predictive value (NPV) 65.2%) and was 38.8%  for non-muscle-invasive low-grade bladder cancer; 75.0%  for non-muscle-invasive high-grade bladder cancer and 68.3%  for muscle-invasive high-grade bladder cancer. The specificity for the statistical calculations was 93.8%. The cut-off value (10 µg/L) was evaluated for the whole patient cohort. The area under the curve of the quantitative UBC® Rapid Test using the optimal threshold obtained by receiver operating characteristics (ROC) analysis was 0.774. Elevated values of UBC®Rapid Test in urine are higher in patients with high-grade bladder cancer in comparison to low-grade tumors and the healthy control group. Conclusions: UBC®Rapid Test has potential to be a clinically valuable urinary protein biomarker for detection of high-grade bladder cancer patients and could be added in the management of NMI-HG tumors. UBC®Rapid results generated in both study centers in the present multicenter study are very similar and reproducible. Furthermore UBC®Rapid Test is standardized and calibrated and thus independent of used batch of test as well as study site.Objectives: UBC In Europe bladder cancer (BCa) is the fifth most frequent cancer. Its incidence rate was 151,200 and its annual mortality rate was 51,400 cases in 2012 [Non-muscle invasive high-grade bladder cancer has a particularly high rate of recurrence and will progress to muscle-invasive disease. The ideal urine-soluble marker should be used for primary diagnosis, follow-up, and screening of high-risk populations; replacing cystoscopy during follow-up or decreasing the number of control cystoscopies during follow-up would be a worthwhile goal. Due to its contact with urine, malignant cells are shed into the urine, and this urine contains the carcinogens producing the malignancy. Some of these urinary based tests have a higher specificity and sensitivity than classical urine cytology and could be important for screening and case findings .Intermediate filaments of the cytoskeleton of epithelial cells containing cytokeratins are often overexpressed in urothelial tumors. In humans twenty different cytokeratins have been identified, and cytokeratins 8, 18, and 19 are known to be important in urothelial cells . The exp®Rapid Test is based upon an immunochromatographic method and measures fragments of cytokeratin 8 and 18 qualitatively. The measured levels are lower in low-grade tumors and benign urological diseases [®Rapid assay with a photometric reader. It is important to highlight that this version of UBC®Rapid Test is a modified and updated version of fast cytokeratin determination in urine in comparison to the assay introduced 15 years ago. Furthermore this new version of UBC®Rapid Test is used in combination with a reader to quantitate the signal quite comparably with an ELISA assay, but it is a point-of-care (POC) assay. Previous UBC Rapid assays were only assays for visual evaluation of results.UBCdiseases . Cytoker®Rapid Test for detecting CIS [®Rapid Test and to evaluate the usefulness of UBC®Rapid Test in patients with urinary bladder cancer with a focus on non-muscle invasive high-grade (NMI-HG) tumors and compare with healthy individuals.In the last publication of our group we had a focus on carcinoma in situ (CIS), and we could show excellent results for UBCting CIS . RegardiA total of 530 patients were included in the study; 242 with confirmed bladder cancer, 62 with non-evidence of disease (NED), and 226 healthy controls with no history of bladder cancer. The median age of the study population was 73 (range 26–98) years. Of these patients, 391 (73.8%) were men and 139 (26.3%) were women. Among the 242 patients with confirmed bladder cancer, 134 had non-muscle-invasive low-grade (NMI-LG), 48 had NMI-HG, and 60 had muscle-invasive high-grade (MI-HG) BCa; 182 (75.2%) had non-muscle-invasive bladder cancer (pTa and pT1 tumors), 60 (24.8%) had stage pT2–4. Carcinoma in situ (CIS) was detected in 23 cases (9.5%). A detailed analysis of the CIS patients in this study had already been published .The number of patients and healthy controls are listed in ®Rapid Test are detectable in urine of bladder cancer patients . The high-risk group showed a markedly higher UBC®Rapid signal than the low-risk group. The area under the curve (AUC) of the quantitative UBC®Rapid Test using the optimal threshold obtained by receiver operating characteristics curve (ROC) analysis (cut-off 10.0 µg/L) was 0.774 as shown in ®Rapid values in boxplots in the different patient groups . It shows also that most of the elevated values are definitely higher than the cut-point, especially for NMI-HG tumors.We could show that elevated concentrations of UBCpatients . Elevate®Rapid specificity was 93.8% for all calculations. Sensitivity was calculated as 38.8% for NMI-LG, 75.0% for NMI-HG, and 68.3% for MI-HG bladder cancer, and the UBC®Rapid Test including the 95% confidence interval are also listed in Data of sensitivity, specificity, positive, and negative predictive values using a cut-off 10.0 µg/L for UBC©Rapid Test are very similar in both centers demonstrating the robustness and stability of UBC®Rapid Test POC assay.The data, which were generated in the two centers separately and reported in ®Rapid Test for diagnosis of bladder cancer with a specific focus on patients with NMI-HG tumors of the urinary bladder compared with healthy individuals. The results of the present study show that cytokeratin concentrations determined by UBC®Rapid Test measured by POC reader are statistically significant for patients with bladder cancer and healthy controls. Values of UBC®Rapid Test in high-grade tumors are significantly higher than in low-grade tumors, NED patients, and healthy individuals. The AUC as a parameter of diagnostic quality was calculated with 0.774. UBC®Rapid Test determined quantitatively could be applied to determine the risk for bladder cancer, but also the risk of having a high-grade tumor with increased risk for recurrence. The need for quantitative urinary markers like UBC®Rapid Test had also been published before [®Rapid Test especially for patients with high-grade bladder cancer [®Rapid, a sensitivity of 65% and a specificity of 92% was calculated [®Rapid with only visual evaluation was available. In our study, the new version of UBC®Rapid Test as cytokeratin assay was used; an improved lateral flow method resulted in a clearer test and control bands to evaluate. In this study UBC®Rapid Test was used with the Omega 100 reader to quantify the results.The main purpose of this multi-center study was to evaluate the clinical usefulness of UBCd before . The resr cancer ,12,13,14lculated ,16. In t®Rapid studies reported a cut-off of 12.3 µg/L, a sensitivity, specificity, PPV, and NPV of 60.7%, 70.1%, 46.8%, and 79.3%, respectively, and with an AUC of 0.68 [®Rapid studies in the literature, the sensitivity of the qualitative UBC®Rapid Test ranged from 46.2% to 78.4%, and its specificity from 82.4% to 97.4% [®Rapid Test POC system has been assessed in just five studies, which reported a sensitivity of 46.6% to 64.5% and a specificity ranging from 70.1% to 86.3%, respectively [©Rapid Test are very similar in both study centers. There is no significant difference in the ROC analyses, showing that the test is very stable and reproducible.Data presented in newer UBCto 97.4% ,18,19,20ectively ,19,20. ICurrently, many different urinary POC test systems are available on the market, permitting non-invasive and rapid determination of urinary markers. Their diagnostic accuracy, however, is mostly controversially discussed in a small number of studies ,22. The ®Rapid Test is an objective method that is standardized and reproducible [According to EAU guidelines, the examination of voided urine to detect cancer cells by cytology has a high sensitivity in high-grade tumors and CIS . The majoducible .The use of those urinary markers for routine follow-up is not recommended in clinical practice by current guidelines and remains a debated issue ,25. The ®Rapid Test was also compared with cytology, showing better results for UBC®Rapid Test [®Rapid with other markers has been shown by Gleichenhagen et al. [®Rapid and survivin increased the sensitivity to 66% with a specificity of 95%. For high-grade tumors, the combination showed a sensitivity of 82% and a specificity of 95%. A combination of both assays confirmed the benefit of using marker panels.It is common that new urine tests are compared with the results of cytology. Across a large number of studies, the results varied a lot. Sensitivity for G1-tumors is lower than 30%, for G2-tumors around 60%, and for G3-tumors 90%. Specificity is around 90–95% . In the pid Test ). In thepid Test . Urinaryn et al. . In this®Rapid Test enables risk stratification for bladder cancer based on the absolute UBC®Rapid Test value. UBC®Rapid Test might not only contribute to improved detection of bladder cancer, but also to improved prediction of high-risk tumors. This has also been shown for other quantitative protein-based urinary tests [®Rapid Test, grade of haematuria, smoking status, age, and gender for developing a nomogram [In contrast to dichotomized urinary tests, the quantitative character of the UBCry tests . An apprnomogram .Of course, there are many other markers on the market, and genetic testing looks especially promising. Regardless, at the moment these markers are a rapid diagnostic tool too complicated to be included into basic and fast diagnostics. Currently, we still must stick to the proteins when we discuss quick testing. However, the “ideal urine-based marker” for detecting bladder cancer recurrence during surveillance would be rapid, non-invasive, and easy to perform and interpret. Furthermore, the assay should possess not only a high specificity to reduce superfluous cystoscopies on oncological follow-up, but also a high sensitivity so that no patient with low-grade and high-grade bladder cancer will be missed .For this prospective study, 530 urine samples from bladder cancer patients and healthy controls have been collected between January 2014 and October 2015 at the Department of Urology, HELIOS, Hospital Bad Saarow (study center 1), and Lukas Hospital Neuss (study center 2), Germany. The study was approved by the local Institutional Review Board of national Medical Association Brandenburg (AS 147(bB)/2013). All patients with confirmed bladder cancer underwent cystoscopy, bladder ultrasound, and transurethral resection of bladder tumor in case of abnormal findings. Exclusion criteria were any kind of mechanical manipulation  within 10 days before urine sampling. Other exclusion criteria were benign prostate enlargement, urolithiasis, other tumor diseases, severe infections, and pregnancy. All these criteria could influence the test to produce false positive results. Less than 10% of the possible study cohort had to be excluded based on exclusion criteria.®Rapid Test . All tests were carried out as recommended by the manufacturer’s instructions. The presence of a test band after 10 minutes of incubation was checked. After visual evaluation, the test cartridges were analyzed by the photometric point-of-care (POC) system Concile Omega 100 reader  for quantitative analysis. The cut-off value used for calculation of statistical parameters was based upon the evaluation of the receiver operating characteristics curve (ROC) and defined as 10.0 µg/L. The Omega 100 reader illuminates the test field with a complementary colored light to reduce interference in the analysis. The built-in charge-coupled device–matrix sensor takes a photograph of the light reflected, which is analyzed by the device. Voided urine samples were collected in a sterile plastic container and subsequently processed. Urine samples were analyzed by the UBChttps://www.R-project.org/). Data are presented descriptively using means and standard deviations for numerical variables and absolute and relative frequencies for categorical variables. Comparison between groups at baseline has been performed using analysis of variance (ANOVA) for numerical variables and chi-square tests for categorical variables. All statistical analyses have been performed using R version 3.2.3 (R Core Team (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL ®Rapid Test measurements to detect bladder cancer was evaluated using Receiver Operating Characteristics (ROC) analysis, where the optimal cut-point was determined using the Youden index [The predictive ability of UBCen index . SensitiThe study was performed according to the Declaration of Helsinki. The study was approved by the local Institutional Review Board of National Medical Association Brandenburg (No. AS 147(bB)/2013 dated by 17 November 2013). Written and informed consent was obtained from each participant.®Rapid Test in urine are higher in patients with non-muscle invasive high-grade bladder cancer in comparison to low-grade tumors and the healthy control group. Sensitivity for non-muscle invasive high-grade tumors is very high with 75% at a specificity of 93.8%. Thus, UBC®Rapid Test has the potential to be a more sensitive and specific urinary protein biomarker to identify patients with high-grade tumors that are difficult to detect in cystoscopy. Results for UBC®Rapid Test in both study centers of the present multicenter study are very similar and reproducible. UBC®Rapid Test is standardized and calibrated, and thus independent of use, batch of test, as well as study site. UBC®Rapid Test should be added in the diagnostics and follow-up for NMI-HG tumors of urinary bladder cancer, though cystoscopy is still an important part of monitoring of bladder cancer.Elevated values of UBC


In vitro cancer 3D models are valuable tools to provide mechanistic insight into solid tumor growth, invasion, and drug delivery. The 3D spheroid model of solid tumors has been the most popular cancer model in use until now. However, previous studies have shown that these spheroid models lack sufficient morphological parameters, which may affect their response to chemicals. In this work, we proposed the fabrication of miniaturized 3D cancer models using collagen type I-based bioprintable bioinks. In the context of a mimicking model for advanced neuroblastoma studies, we showed that cancer cells contained in bioprintable bioinks formed Homer Wright-like rosettes, maintained their proliferative capacities and produced an equivalent Vimentin-rich matrix unlike that of non-bioprintable bioinks which made for poorer models. In addition, bioprintable bioinks were successfully bioprinted as compartmentalized 3D models in the centimeter scale, which was not feasible using non-bioprintable bioinks. In contrast to non-bioprintable hydrogels, we did not observe contraction in their bioprintable counterparts, which is an advantage for prospective 3D bioprinted models that should attain stable rheological and mechanical properties after bioprinting. By adopting this proposed system for the use of patient-derived primary tumor cells, the approach could be introduced as a first line strategy in precision medicine for testing the response of neuroblastoma cells to drugs, especially when disease progresses rapidly or patients do not respond to actual therapy regimens. In vitro cancer 3D models are valuable tools to provide mechanistic insight into solid tumor growth, invasion and drug delivery. They are slowly replacing the use of conventional 2D in vitro cell culture as the main method of adherent cell growth that has been practiced for decades in determining complex cellular and biological processes that occur during tumorigenesis. Advances in 3D cell culture technologies, biomaterials science, and biofabrication have enabled improvements in the development of in vitro cancer models. In recent years, spheroid-based models, hybrid models, and tumor–microvessel models have been studied and characterized . The 3D Neuroblastoma is the most common extracranial malignancy that occurs in preschool children, although occasionally it can be diagnosed in adolescents and adults . Upon diWith the advance of biofabrication strategies, in vitro 3D organotypic constructs have been created for a myriad of applications, ranging from bone and cartilage substitutes to cancer models ,16. In tAgarose and collagen type I stock solutions were prepared as previously described ,19. BrieOscillatory tests were performed in a rotational rheometer  using a 4° rotational cone-plate . Non-bioprintable (0.3% COL) and bioprintable bioinks (0.5% AG-0.2% COL) were measured both in liquid and in gelled form. Rheological analysis of liquid gels was performed at 37 °C and polymerized gels were analyzed at 25 °C after gelation of the hydrogels. A 5-step oscillatory sequence was performed for each of the hydrogels in both liquid and gelled form, based on previously published protocols : (1) fixALK gene mutation F1174L, specific for SH-SY5Y cells  human primary umbilical cord-derived mesenchymal stromal cells , (2) human primary umbilical vein endothelial cells (HUVEC), and (3) human bone marrow-derived epithelial-neuroblastoma immortalized cells (SH-SY5Y). Human primary MSC and HUVEC were isolated according to previously published protocols ,22. MSC 5Y cells , by PCR lsewhere .6 cells per mLMSC were loaded at 106 cells per mLHUVEC were loaded at 3 × 106 cells per mLSH-SY5Y were loaded at 10Single, co-, and tricultures using MSC, HUVEC, and SH-SY5Y were prepared using bioprintable and non-bioprintable hydrogels. For non-bioprintable hydrogels, we used collagen type I hydrogel matrices. For bioprintable hydrogels, we used agarose-collagen type I blends. Final cell concentrations of single, co-, and tricultures were the same for both bioprintable and non-bioprintable hydrogels:Cell number was determined using trypan-blue exclusion assay and Countess™ automated cell counter . Single cell cultures of MSC, HUVEC and SH-SY5Y were used as control cultures for the three cell types. In these samples cells were cultivated in their respective media . Single cultures in non-bioprintable hydrogels were prepared by mixing MSC, HUVEC, or SH-SY5Y in collagen type I hydrogel with a final concentration of 0.3%. Single cultures in bioprintable hydrogels were prepared by mixing the cells in agarose-collagen type I blends with a final concentration of 0.5% and 0.2%, respectively, for agarose and collagen. Cell-loaded hydrogels were casted with the respective cell densities for each cell type and polymerized at 37 °C for 30 min (non-bioprintable samples) or 1 min at 25 °C (bioprintable samples). Samples were incubated in their respective culture media at 37 °C for up to 14 days. One additional single culture of SH-SY5Y in EBM-2 was added to the experimental set-up for excluding possible differences with the single culture in DMEM.Co-cultures of SH-SY5Y/MSC and SH-SY5Y/HUVEC were prepared with both non-bioprintable and bioprintable hydrogels and cultivated in endothelial medium (EGM-2). The final hydrogel concentrations and cell densities were the same as used for single cultures. The polymerization and culture conditions of co-cultures were the same as for single cultures, with the exception of the culture medium (EGM-2).Tricultures of SH-SY5Y/MSC/HUVEC were prepared with both non-bioprintable and bioprintable hydrogels and cultivated in EBM-2. The final hydrogel concentrations and cell densities were the same as used for single and co-cultures. The polymerization and culture conditions of tricultures were the same as for co-cultures.Macroscopic appearance and contraction of cell-loaded hydrogels after in vitro culture was recorded using a photographic camera .Cell-loaded hydrogel samples were histologically evaluated after in vitro culture. After 14 days of incubation, samples were fixed in 4% formaldehyde for 2 h, transferred to 70% ethanol, and dehydrated overnight. Then, samples were embedded in paraffin, cut into 8-µm slices, and stained histologically with hematoxylin and eosin (HE). Immunohistological staining was performed with Ki67  and vimentin . Patients’ derived material was analyzed at the University Hospital of Padua, Italy. The tumor masses were examined under a light microscope  with a Nikon Coolpix camera under 10× and 20× magnification.SH-SY5Y used in this study were stably expressing GFP. The imaging of SH-SY5Y distribution in the hydrogels was performed after DAPI co-staining . Samples were imaged with a two-photon laser scanning microscope  mounted with a 25× NA 1.05 water dipping objective. Excitation wavelength was mode-locked to 800 nm using a pulsed Ti:Sapphire laser . Single images were recorded in the XY axis and collected at 1 µm z-stack intervals. In order to visualize human MSC and HUVEC, samples were stained after fixation in 4% formaldehyde with phalloidin-TRITC for labelling F-actin  and DAPI for nuclear staining .Cell-free 0.5% AG-0.2% COL bioprintable bioink was printed with a resembling architecture as proposed in The creation of 3D cancer models using drop-on-demand bioprinting involves the use of hydrogels, which are not only biocompatible, but also succeed in mimicking in vitro microenvironments. In this work, we studied the printability of 0.2% COL-0.5% AG as bioinks for creating 3D models as well as the interaction of cancer cells with stromal and endothelial cells in non-bioprintable hydrogels as control matrices (0.3% COL) and in bioprintable hydrogels in light of their usability for bioprinting. A 3D model consisting of three distinct parts  was designed in CAD and sliced a,b. FiguCancer tissue that is formed in the human body consists not only of cancer cells, but also of other cell types surrounding the tumor. Stromal cancers like neuroblastoma are characterized by the presence of stromal cells like Schwann cells and can be stroma-rich or stroma-poor . Thus, iAs a proof-of-principle, the 3D cancer model proposed in this work should ideally resemble the microenvironment of native neuroblastoma cancer. It is well-known that neuroblastoma rosettes can be found in histological and aspirated bone marrow smears . NeuroblAdvanced neuroblastoma cancer cells are highly proliferative cells in vivo and in vitro . NeverthThe proposed theoretical 3D cancer model  was compIn this work, we proposed the fabrication of miniaturized 3D cancer models using collagen type I-based bioprintable bioinks. In the context of a mimicking model for advanced neuroblastoma studies, we showed that cancer cells contained in bioprintable bioinks formed Homer Wright-like rosettes, maintained their proliferative capacities and produced Vimentin-rich matrices. In addition, the presence of MSC triggered the production of elastic matrix, which consequently forced non-bioprintable hydrogels to contract. In contrast, we could not observe contraction in bioprintable hydrogels, which is an advantage for prospective 3D bioprinted models that should attain stable rheological and mechanical properties after bioprinting. Moreover, vascular tube formation in the presence of cancer cells was not observable neither in non-bioprintable hydrogels nor in bioprintable bioinks. A possible explanation for this is the small size of the generated tumoroids which may offset the need for such structures given that nutrients can easily reach tumor cells by diffusion. On the contrary, it is known from the literature and has been confirmed in our lab that the 3D co-culture of HUVEC and MSC, in the absence of cancer cells, leads to a production of regular and well-defined microvasculature, implying a more cooperative dynamic between these two cell types. Thus, to avoid the described limitation met during the histological examination of our models, we proposed a novel design for 3D stromal tumoroids, in which the vasculature should contain only HUVEC, or a combination of HUVEC and MSC, in order to achieve a successful route for nutrients to reach the resting cells of the developed bioprintable model. In this manner, by adopting bioprinting methodology we could guarantee a uniform and more controlled way of generating the 3D tumoroids that can subsequently be used for cell biology, molecular or pharmaceutical studies in a median-throughput range. In addition, by adopting this system for the use of patient-derived primary tumor cells, the approach could be introduced as a first line therapy in a precision medicine approach to testing the response of neuroblastoma cells to drugs, especially when disease progresses rapidly, or patients do not respond to actual therapy regimens.